Can I use PBS instead of DPBS?

It depends on the experiment. If your protocol requires calcium and magnesium (e.g., for cell adhesion assays), DPBS with Ca²⁺/Mg²⁺ is not interchangeable with standard PBS, which lacks these ions. For simple washing steps where divalent cations are not critical, PBS and DPBS without Ca²⁺/Mg²⁺ are functionally equivalent.

Why does HBSS contain glucose but PBS does not?

HBSS was designed as a balanced salt solution that can sustain cells for short-term incubations outside a CO₂ incubator. The glucose (5.6 mM) serves as an energy source. PBS is a simpler buffering system intended primarily for washing and dilution, not for maintaining cell metabolism.

Which buffer should I use for flow cytometry staining?

Most flow cytometry protocols use PBS or DPBS (without Ca²⁺/Mg²⁺) as the base for staining and wash buffers. FACS buffer is typically PBS + 2% FBS + 0.1% sodium azide. HBSS is less common for flow cytometry but can be used if your protocol specifies it.

Do I need to filter-sterilize these buffers?

For cell culture applications, yes — all three buffers should be sterile-filtered (0.22 µm) or autoclaved (PBS only; autoclaving HBSS or DPBS containing glucose or calcium can cause precipitation). Commercial pre-made sterile versions are also available.

Can I autoclave DPBS or HBSS?

Standard PBS can be autoclaved safely. However, DPBS with Ca²⁺/Mg²⁺ and HBSS containing glucose should be sterile-filtered (0.22 µm) rather than autoclaved, as autoclaving can cause calcium phosphate precipitation and glucose degradation.

PBS vs DPBS vs HBSS: Which Buffer Should You Use?

Introduction

Choosing the right buffer is one of those deceptively simple decisions that can make or break an experiment. PBS, DPBS, and HBSS are the three most common isotonic buffers in cell biology labs, yet researchers often use them interchangeably — sometimes without realizing the consequences.

This guide breaks down the composition, purpose, and practical considerations for each buffer so you can pick the right one every time.

Quick Comparison Table

Property	PBS	DPBS	HBSS
Full name	Phosphate Buffered Saline	Dulbecco's Phosphate Buffered Saline	Hank's Balanced Salt Solution
pH	7.4	7.4	7.0–7.4
Osmolarity	~280 mOsm	~280 mOsm	~290 mOsm
Ca²⁺	✗	Optional (0.9 mM)	Optional (1.3 mM)
Mg²⁺	✗	Optional (0.5 mM)	Optional (0.9 mM)
Glucose	✗	✗	✓ (5.6 mM)
Bicarbonate	✗	✗	✓ (4.2 mM)
Buffering	Phosphate only	Phosphate only	Phosphate + Bicarbonate
Best for	Washing, dilution	Cell culture washing	Short-term cell incubation

PBS (Phosphate Buffered Saline)

Composition

Standard 1× PBS contains:

NaCl — 137 mM (ionic strength)
KCl — 2.7 mM (ionic strength)
Na₂HPO₄ — 10 mM (buffering)
KH₂PO₄ — 1.8 mM (buffering)

Key Properties

PBS is the simplest of the three buffers. It maintains physiological pH (7.4) and osmolarity (~280 mOsm/L) using only phosphate salts and sodium/potassium chloride. No divalent cations, no glucose, no bicarbonate.

When to Use PBS

General washing of cells, tissues, or blots
Diluting antibodies for Western blot, ELISA, or immunostaining
Resuspending cell pellets for analysis (not culture)
Immunohistochemistry and immunofluorescence rinses
Any situation where you need an isotonic buffer and divalent cations would interfere

When NOT to Use PBS

Cell culture washing before trypsinization — PBS works, but if your cells are sensitive, the lack of Ca²⁺/Mg²⁺ may cause premature detachment
Protocols requiring glucose for metabolic support
Long-term cell incubation outside a CO₂ incubator (no buffering against CO₂ loss)

DPBS (Dulbecco's Phosphate Buffered Saline)

Composition

DPBS comes in two formulations:

DPBS without Ca²⁺/Mg²⁺:

NaCl — 137 mM
KCl — 2.7 mM
Na₂HPO₄ — 8.1 mM
KH₂PO₄ — 1.5 mM

DPBS with Ca²⁺/Mg²⁺:

All of the above, plus:
CaCl₂ — 0.9 mM
MgCl₂ — 0.5 mM

Key Properties

DPBS was developed by Renato Dulbecco as a modification of PBS for use with cell cultures. The phosphate concentrations are slightly lower than in standard PBS, and the "with Ca²⁺/Mg²⁺" version adds divalent cations that are important for cell adhesion and signaling.

When to Use DPBS

Cell culture washing — DPBS (–Ca²⁺/–Mg²⁺) is the standard wash before trypsinization, because removing divalent cations helps trypsin detach cells
Cell culture washing where adhesion matters — DPBS (+Ca²⁺/+Mg²⁺) when you want to wash without detaching adherent cells
Live-cell experiments where you need a cleaner buffer than PBS but don't need glucose
Dissociation protocols — Ca²⁺-free DPBS is preferred over PBS in many cell dissociation protocols

DPBS (–) vs DPBS (+): The Critical Difference

This is the most common source of confusion:

DPBS (–Ca²⁺/–Mg²⁺): Promotes cell detachment. Use before trypsinization or enzymatic dissociation.
DPBS (+Ca²⁺/+Mg²⁺): Supports cell adhesion. Use for gentle washing when you want cells to stay attached.

Always check your protocol. Using the wrong variant can ruin your experiment.

HBSS (Hank's Balanced Salt Solution)

Composition

HBSS is the most complex of the three:

NaCl — 137 mM
KCl — 5.4 mM
Na₂HPO₄ — 0.3 mM
KH₂PO₄ — 0.4 mM
NaHCO₃ — 4.2 mM
D-Glucose — 5.6 mM
CaCl₂ — 1.3 mM (optional)
MgSO₄ — 0.8 mM (optional)

Key Properties

HBSS was designed by John Hanks in 1940 as a more physiologically complete medium. It includes glucose for metabolic energy and bicarbonate for pH buffering in a CO₂ environment. This makes it suitable for short-term incubations where cells need more than just osmotic support.

When to Use HBSS

Short-term cell incubation outside complete media (up to a few hours)
Cell-based assays that require metabolically active cells (e.g., uptake assays, calcium imaging)
Tissue dissociation and primary cell isolation
Organ perfusion and tissue transport from surgery to lab
Calcium imaging — HBSS provides a good baseline for dye-loading and imaging

When NOT to Use HBSS

Simple washing — HBSS is overkill; use PBS or DPBS instead
Long-term culture — HBSS lacks amino acids, vitamins, and serum; it cannot replace complete media
Autoclaving — The glucose in HBSS can caramelize; use sterile filtration instead

Decision Guide: Which Buffer to Choose

Start with these questions:

Are you just washing or diluting? → PBS (simplest, cheapest, most versatile)
Are you washing cultured cells before trypsinization? → DPBS (–Ca²⁺/–Mg²⁺)
Are you washing cultured cells but need them to stay attached? → DPBS (+Ca²⁺/+Mg²⁺)
Do your cells need metabolic support for the next few hours? → HBSS
Are you doing calcium imaging or ion-sensitive assays? → HBSS (with or without Ca²⁺ depending on assay)
Are you isolating primary cells or dissociating tissue? → HBSS (with glucose for cell viability)

Common Protocol-Buffer Pairings

Protocol	Recommended Buffer
Western blot washing	PBS or PBST
ELISA washing	PBS or PBST
Immunofluorescence wash	PBS
Cell culture wash (pre-trypsin)	DPBS (–Ca²⁺/–Mg²⁺)
Cell culture wash (gentle)	DPBS (+Ca²⁺/+Mg²⁺)
Flow cytometry staining	PBS (as FACS buffer base)
Calcium imaging	HBSS
Primary cell isolation	HBSS
Drug treatment incubation	HBSS or serum-free media
Tissue transport	HBSS (cold, on ice)

Common Mistakes to Avoid

1. Using PBS when the protocol says DPBS

PBS and DPBS have slightly different phosphate concentrations. For most applications this doesn't matter, but for sensitive cell-based assays, use exactly what the protocol specifies.

2. Forgetting to check the Ca²⁺/Mg²⁺ status

The most consequential mistake. Using DPBS (+Ca²⁺/+Mg²⁺) before trypsinization will inhibit trypsin activity and reduce cell yield. Using DPBS (–Ca²⁺/–Mg²⁺) for a gentle wash may cause cells to detach.

3. Autoclaving HBSS

The glucose in HBSS degrades and caramelizes at autoclave temperatures. Always sterile-filter HBSS through a 0.22 µm filter.

4. Storing buffers at wrong temperature

PBS: Room temperature or 4°C; stable for months
DPBS with Ca²⁺/Mg²⁺: 4°C; check for precipitates before use
HBSS: 4°C; use within recommended shelf life as glucose can degrade

5. Using expired or contaminated buffer

Always check for turbidity, color changes, or particulates. When in doubt, make a fresh batch.

Preparation Tips

All three buffers are available commercially as ready-to-use solutions or as 10× concentrates. For labs that go through large volumes, preparing from powder or individual salts is more cost-effective.

Use MakeSolution's free calculator to get exact amounts for any volume — just type "500 mL PBS", "1 L DPBS", or "HBSS 250 mL" and get a complete preparation protocol with step-by-step instructions.

Summary

If you need...	Use...
A simple, cheap wash buffer	PBS
Cell culture washes (general)	DPBS (–Ca²⁺/–Mg²⁺)
Gentle cell washes (keep adhesion)	DPBS (+Ca²⁺/+Mg²⁺)
Short-term cell incubation	HBSS
Metabolic support during assays	HBSS
Tissue dissociation/transport	HBSS

The bottom line: PBS is your workhorse, DPBS is your cell culture companion, and HBSS is your go-to when cells need more than just salt water.